The UC is one of the first structures to form following gastrulation (formation of the three embryonic germ layers). As folding is initiated, the embryonic disc becomes connected, by the primitive midgut (embryonic origin) to the primitive yolk sac (extra-embryonic origin) via the vitelline and allantoic vessels which in turn develop to form the umbilical vessels (Haynesworth et al., 1998; Pereda and Motta, 2002; Tuchmann-Duplessis et al., 1972). These vessels are supported in, and surrounded by, what is generally considered a primitive mesenchymal tissue of primarily extra-embryonic derivation called Wharton's Jelly (WJ) (Weiss, 1983). From this early stage, the UC grows, during gestation, to become the 30-50 cm cord seen at birth. It can be expected therefore, that WJ contains not only the fibroblast-like, or myo-fibroblast-like cells which have been described in the literature (see below), but also populations of progenitor cells which can give rise to the cells of the expanding volume of WJ necessary to support the growth of the cord during embryonic and fetal development.
WJ was first described by Thomas Wharton, who published his treatise Adenographia in (1656) (Wharton T W. Adenographia. Translated by Freer S. (1996). Oxford, U.K.: Oxford University Press, 1656; 242-248). It has subsequently been defined as a gelatinous, loose mucous connective tissue composed of cells dispersed in an amorphous ground substance composed of proteoglycans, including hyaluronic acid (Schoenberg et al., 1960), and different types of collagens (Nanaev et al., 1997). The cells dispersed in the matrix have been described as “fibroblast-like” that are stellate in shape in collapsed cord and elongate in distended cord (Parry, 1970). Smooth muscle cells were initially observed within the matrix (Chacko and Reynolds, 1954), although this was disputed by Parry (1970) who described them as somewhat “unusual fibroblasts” which superficially resemble smooth muscle cells. Thereafter, little work had been done on characterizing these cells until 1993 when Takechi et al. (1993) performed immunohistochemical investigations on these cells. They described the cells as “fibroblast-like” that were “fusiform or stellate in shape with long cytoplasmic processes and a wavy network of collagen fibres in an amorphous ground substance” (Takechi et al., 1993). For the immunohistochemical staining, they used primary antibodies against actin and myosin (cytoplasmic contractile proteins), vimentin (characteristic of fibroblasts of embryonic mesenchyme origin) and desmin (specific to cells of myogenic origin) in order to determine which types of myosin are associated with the WJ fibroblasts. They observed high levels of chemically extractable actomyosin; and although fibroblasts contain cytoplasmic actomyosin, they do not stain for actin or myosin, whereas the WJ fibroblasts stained positively for both. Additionally, positive stains for both vimentin and desmin were observed leading to the conclusion that these modified fibroblasts in WJ were derived from primitive mesenchymal tissue (Takechi et al., 1993). A subsequent, more recent study by Nanaev et al. (1997) demonstrated five steps of differentiation of proliferating mesenchymal progenitor cells in pre-term cords. Their findings supported the suggestion that myofibroblasts exist within the WJ matrix. The immunohistochemical characterization of the cells of WJ, shows remarkable similarities to that of pericytes which are known to be a major source of osteogenic cells in bone morphogenesis and can also form bone nodules referred to as colony forming unit-osteoblasts (CFU-O) (Aubin, 1998) in culture (Canfield et al., 2000).
Recent publications have reported methods to harvest cells from UC, rather than UC blood. Mitchell et al. (Mitchell et al., 2003) describe a method in which they first remove and discard the umbilical vessels to harvest the remaining tissue. The latter, which will include both the remaining WJ (some of which will have been discarded with the vessels, since the umbilical vessels are entirely enveloped in WJ) and the amniotic epithelium, is then diced to produce small tissue fragments that are transferred to tissue culture plates. These tissue fragments are then used as primary explants from which cells migrate onto the culture substratum.
In another publication, Romanov et al. (2003) indicate they were successful in isolating mesenchymal stem cell-like cells from cord vasculature, although they also indicate their cultures do not contain cells from WJ. Specifically, they employ a single, 15 min, collagenase digestion from within the umbilical vein, which yields a mixed population of vascular endothelial and sub-endothelial cells. Romanov et al. show that sparse numbers of fibroblast-like cells appear from this cell harvest after 7 days.
Also, U.S. Pat. No. 5,919,702 describes a method of isolating “pre-chondrocytes” from the WJ of human UC, and their use to produce cartilage. Particularly, the method comprises slicing open a one inch section of cord longitudinally, dissecting away the blood vessels and ‘casing’, which are then discarded, and collecting the WJ into a sterile container where it was cut into 2-3 mm3 sections for culturing. In a preferred method, cells are isolated by placing a 2-3 mm3 section of the WJ on a glass slide on the bottom of a Petri dish, covering it with another slide, and culturing it for 10-12 days in order to allow the ‘pre-chondrocytes’ to migrate out to the culture dish surface.
In US patent application US2005/0148074 published Jul. 7, 2005, Davies et al describe the isolation of a unique progenitor cell population from a particular region of Wharton's jelly, termed the perivascular region. The Wharton's jelly in this region is on or associated with the external walls of the cord vessels, and remains associated with the vessels when they are excised from the cord. The progenitor cell population has a remarkably short doubling time, and comprises progenitor cells having a wide range of valuable properties, including cells that are multipotent and give rise to various mesenchymal tissues including fat, bone, cartilage, muscle and endothelium; cells that spontaneously differentiate into bone-forming osteoblasts; and cells that lack MHC class I and class II markers. Progenitor cells obtained from the Wharton's jelly in the perivascular region of human umbilical cord vasculature are referred to herein as HUCPVCs.
Cord tissue thus promises to be an important source of progenitor and other cells for use in tissue engineering and other medical procedures. Current practices permit the longer term storage of cord-derived cells under cryogenic conditions, and the recovery of viable cells therefrom. However, techniques have yet to be developed that permit the recovery of viable cells from frozen or cryogenically stored cord tissue, as distinct from cord cells per se. Accordingly, where the recovery of viable cells from cord tissue is required, technicians are required to act promptly to process cord tissue while it is fresh, usually within 24 hours from cord extraction, so that the desired tissue can be extracted, and the desired cells isolated, cultured and stored frozen, while those cells are still viable. Clearly, it would be useful to provide a method permitting the cord tissue to be stored, to serve as an on-demand source of viable cells.